A cell-free system comprising liver mitochondria incubated in a physiological environment with respect to soluble ionic and nucleotide components, and of energetic and respiratory steady-state has been developed for a unique approach to studies of metabolic regulation. This system has been designed for use in conjunction with soluble cellular enzymes to evaluate (a) control of the flux of pyruvate carbon to acetyl-CoA, ketone bodies, anaplerotic reactions into the citric acid cycle, and synthesis and flux of pyruvate-derived intermediates into glucose- and fatty acid- precursors; (b) factors which may regulate the capacity of the pathway for ketogenesis; and (c) concerted interactions between glycolytic systems, their co-factors and transmembrane fluxes of substrates (and reducing equivalents which move as "passengers" with substrate movements) and the process of oxidative phosphorylation. These investigations will all be carried out under steady-state energetic conditions with respect to phosphorylation and reduction potentials (ATP/ADP x phosphate and NAD ion/NADH ratios respectively) over the ranges which may obtain over the extremes of physiological and pathological conditions. Functionally isolated perfused rat leg muscle preparations will be used for continuing studies involving the identification and quantitation of pathways responsible for anaplerotic maintenance of, and net removal of carbon from the citric acid cycle (i.e., flux and homeostasis). Specifically these studies will be directed toward identifying the source of carbon for, and pathways involved in (a) synthesis of net cycle intermediates promoted by fatty acids and ketone bodies; (b) synthesis of alanine which is released from muscle during periods of negative nitrogen balance; and (c) synthesis of cycle intermediates resulting from acute, intense contraction. The NAD-linked mitochondrial malic enzyme will be isolated from heart and skeletal muscle and compared on the basis of physical and kinetic properties.